1028
Resonance Raman Study of Cobalt( 11)-Imidazole Complexes as Models for Metalloproteins' Calvin M. Yoshida, Teresa B. Freedman, and Thomas M. Loehr* Contributionfrom the Department of Chemistry, Oregon Graduate Center, Beaverton. Oregon 97005. Received July 18, 1974
Abstract: The Raman spectra of cobalt(I1)-imidazole complexes of known stoichiometries and geometries are reported, using Ar+ laser excitation. The excitation frequency dependence of Raman peak intensities, which differs for octahedrallyand tetrahedrally-coordinated complexes, indicates a slight resonance of the vibrations with the 4Tl,(F) 4T2g(F)Co(I1) d-d transition in the octahedral complex, and with a uv charge-transfer state in the tetrahedral complexes. In the latter complexes, the more pronounced resonance effect of the ring vibrations supports assignment of a number of peaks to ring modes rather than C-H vibrations. Resonance enhancement in the tetrahedrally-coordinated Co(I1)-histidine complex is very minor. The low Raman intensity and only slight resonance effects observed in the model complexes make it unlikely that Co(I1)-substituted zinc proteins would exhibit a resonance-enhanced Raman spectrum of the cobalt-protein chromophore.
-
In resonance Raman (rR) spectra2 of metalloproteins only those molecular vibrations associated with the metalligand chromophore are observed to undergo intensity enhancement. In the case of heme protein^^-^ laser excitation wavelengths near the a and ,6 absorption bands of the protein produce a different set.of resonance-enhanced Raman peaks than excitations near the y band. The resonance-enhanced peaks are assigned to vibrations of the porphyrin macrocycle. The internal vibrations of ligands attached at the 5 and 6 positions of the central iron atom of the heme are not observed since the orbitals of these ligands are not involved in electronic transitions giving rise to a , p, or y absorption bands. In the case of iron-sulfur proteins, the rR spectra reveal principally the Fe-S vibrations of cysteine and inorganic sulfur ligand^.^,^ Cobalt(I1)-substituted zinc enzymes are potentially suitable for rR spectroscopic studies because these enzymes possess visible absorption spectra due to the Co(I1)-protein complex, whereas the native Zn enzymes are colorless. Cobalt has been substituted for zinc in the enzymes carbonic anhydrase,8 alkaline p h o ~ p h a t a s e and , ~ carboxypeptidase1° with little or no loss in catalytic activity. The amino acids which have been implicated as metal ligands in the Zn-containing enzymes are two histidines and one glutamic acid in carboxypeptidase' and thermolysin12 and three histidines in carbonic anhydrase.13 Since histidine is the predominant ligand, any rR spectrum of a Co(I1)-substituted zinc protein should be principally due to the Co(I1)-histidine chromophore. We have investigated the Raman spectral properties of Co(I1)-imidazole and -histidine complexes of known stoichiometries and geometries to determine those ligand vibrations that undergo rR enhancement.
Experimental Section Histidine (Sigma), imidazole (J. T. Baker), and the metal salts (Mallinckrodt) were of the highest purity availablz from the respective suppliers and were used without further purification. Cobalt(I1) complexes with imidazole, C3H4N2 (Im), and imidazolate anion, C3H3N2- (Im'), were prepared as f01lows:'~ C0~~ImzC12and CO"h&(N03)2 from C o C 1 ~ 6 H 2 0 and Co(N03)2.6HzO, respectively, with imidazole in neutral ethanol solution and precipitated by the addition of ether; Co111m'2 from CoC12. 6 H 2 0 and imidazole in ethanol with sufficient ethanolic KOH added to precipitate the imidazolate complex. Bis complexes of cobalt(I1) with histidine, C6Hg02N3 (His), and histidinate anion, C6H802N3- (His'), were prepared,I5 respectively, from CoS04-7H20 and histidine in neutral aqueous solution or with sufficient added NaOH to produce the purple histidinate complex. Solids were isolated by evaporating to dryness in a
Journal of the American Chemical Society
rotary evaporator. All steps were carried out in a glove box purged with chromous perchlorate scrubbed Nz, Ultraviolet spectra were recorded on a Cary 16 spectrophotometer. All Raman spectra were recorded on polycrystalline samples mixed with KCI as a bulk carrier and with either K2SO4 or NO3as internal intensity stzndards. Mixtures were lightly pressed into a 5 cm diameter disk and spun a t 2000 rpm. A Jarrell-Ash 25-300 Raman spectrometer with a Coherent Radiation Laboratories Model 5 2 Ar+ laser was used. No sample decomposition was observed even after several hours of continuous irradiation with up to 750 mW of power at the sample. Spike filters were used to eliminate laser plasma lines. Intensity data are based on peak height measurements, and ratioed to the q(A1) vibration of S042-. For Co1'Im6(NO3)2 the vl(A1) vibration of NO3- served as the internal standard. Since we observed a slight (-10%) increase of IU,(~~p-)/I,,,(~ with 0 1 2decreasing -) wavelength of excitation in KN03-K2S04 mixtures, the Co"Irn~(NO3)zdata are corrected to sulfate.
Results The Raman spectrum of Co111mg(N03)2 (Figure l a ) is nearly identical with that of Co"Im2C12 and both are very similar to the spectrum of free imidazoleI6 (Table I). Some dissimilarities between free and complexed imidazole are observed in the 1075-1175-cm-' region. CoIIIm'2 has a Raman spectrum resembling that of the free imidazolate anion (Table I, Figure Ib). At neutral or slightly acidic pH, CoIIHisz forms a pinkcolored complex characteristic of octahedral coordination. The reactivity of this material toward atmospheric oxygen, forming a brown Co(II1)-peroxo complex,'' prevented us from obtaining its Raman spectrum. In strongly basic solutions, however, the deep violet Co'IHHis'2 complex having tetrahedral coordination is formed.15 This complex is sufficiently stable in air to permit recording of its Raman spectrum. While the overall spectrum is weak, the predominant peaks between 1000 and 1600 cm-l with 476.5-nm excitation appear at: 1002 (w), 1063 (w), 1082 (m), 1172 (m), 1210 (m), 1260 (s), 1282 (w), 1308 (m), 1335 (m), 1391 (w), 1425 (w), 1470 (w), 1489 (w), and 1559 cm-' (w). The intensities of strong peaks in the spectra of the complexes were measured relative to the intensity of the internal standard and are plotted against excitation wavelength in Figures 2, 3, and 4. These relative intensities, normalized to the relative spectral intensities using one of the excitation wavelengths, are listed in Tables 11,111, and IV. Discussion Vibrational Assignments. The elemental analyses reported for the complexes, Co1I1m2C12 and C O ~ ~ I ~ ~ ( N O ~ ) ~ ,
/ 97:5 / March 5, 1975
1029 Table I. Raman Spectral Frequencies (cm-l) of Imidazoles, Co(I1) Complexes of Imidazoles, and Spectral Assignments between 900 and 1550 cm-l Ima
CoxxImnC1,h
1532w
1534 w
1486 m 1430 m 1328 s
1495 w 1421 m 1350 w 1322m
1258 s 1160m 1130m
1253 s 1129 w 1165s
1095 w 1066 w
ltO0w
Co111ms(N03)2b
CoIIIm'?b
Im'c
HIm+ d
Assignmentse
~
1540 w 1517 w 1495 w 1439 m 1331 m 1258 s 1147 m 1173 s 1134 w 1105 m 1068 w
950 w 913 w
915vw
Ring 1460 s
1489 m
1457 m
Ring Ring
1332 vw 1313 w 1273 s
1330 w 1307 w 1249 s
1215s
1163 s
1143 s
1135 w
Ring 6(N-H) Ring
1103 w 1085 w 950 vw
llO0w 1075 w 945 w
1106m 1068 vw
6(C-H) 6(C-H)
913 w
6(C-H)
Ring
920 w
Aqueous solution of imidazole, C3H4N2,514.5-nm excitation. * Co(I1) complex, polycrystalline, 457.9-nm excitation. Alkaline solution (-3 M KOH) of imidazole, 476.5 nm. Acidic solution (-6 M HC1) of imidazole, 476.5 nm. e 6(C-H) and 6(N-H) assignments from ref 16 and 18; ring assignments from ref 16,18, and this work.
>
f
1.0
5v
-
z W
c
z
5.
W a , ,
I
2 I-
i
l
d
-
0.1 K
0 450
470
_I
490
510
WAVELENGTH (NM)
I 1700
-
a
1500
1300
I IO0
1 900
Frequency cm-l Figure 1. Raman spectra of cobalt(I1)-imidazole complexes with 457.9-nm excitation: (a) Co"Im~(NO3)2(s), peak at 1045 cm-I is V I (N03-); (b) Co"Im'2 (s), peak at 980 cm-' is V I (SOa2-).
show that one nitrogen in the imidazole is protonated as in the free ligand.I4 Upon complexation of the imidazole to Co2+ through the other nitrogen, the symmetry (C,) of the ligand is not altered. For very strong ligand-cobalt coordination, one would expect that the Raman spectra of the complexes would resemble that of the imidazolium cation (Him'). However, the similarity of the observed spectra to that of free imidazole implies that the ligand is only weakly coordinated to the metal ion. A comparison of the 1075-1175-~m-~regions of the spectra of the free and complexed imidazoles suggests that the 6(C-H) vibration of free imidazole assigned at 1095 cm-' l 6 , I 8 is shifted to -1105 cm-* in the complexes. The moderately intense 1160 cm-' 6(N-H) and 1130 cm-l (ring) vibrations of free imidazole are apparently shifted to 1147 and 1 173 cm-l, respectively, based on the resonance intensity behavior (see below). There is disagreement in the literature concerning the assignment of the two most intense peaks in the Raman spectrum of imidazole. The 1258-cm-' peak has been assigned to a 6(C-H) by Perchard, et al., from infrared and Raman studiesi6 and to a ring mode by Cordes and Walter from a
Figure 2. Dependence of Raman peak intensities of CoI1Img(N03)2 on excitation wavelength: ( 0 ) 920; (m) 1105: (A) 1173; (V) 1258: (e) 1331 cm-I I
0' 450
470
490
WAVELENGTH
510
(NM)
Figure 3. Dependence of Raman peak intensities of Co"Im2C12 on excitation wavelength: ( 0 )913;).( 1100; (A) 1165; (V) 1253; (*) 1322 cm-' .
normal coordinate treatment of ir data.'* On the other hand, the 1331-cm-l peak has been assigned to a 6(C-H) mode by Cordes and Walter and to a ring mode by Per-
Loehr et al.
/
Resonance Raman of Cobalt(I1) Imidazole Complexes
1030 Table 11. Raman Intensities of Co1IIm8(N0& (Relative to the Internal Standard) Normalized to the Relative Intensities using the 457.9-nm Excitation Wavelength. and the Calculated Relative Intensity Enhancement. F.h* (eq 1)
I Raman freq, cm-l
I
W
S
1540 1439 1331 1258 1173 1147 1105 920 Calcd for 1000 cm-1, 1060 nm
I
vel
0'
450
470
510
490
WAVELENGTH
(NM)
Figure 4. Dependence of Raman peak intensities of Co"His'2 on excitation wavelength: ( 0 )1180; (m) 1218; (V) 1267 cm-'.
chard, et al. Since the 133I-cm-I peak decreases considerably in intensity upon conversion of imidazole to imidazolate anion but is unaffected by deuteration of the nitrogen in free imidazole,I6 it is more likely that the most significant contribution to this peak is from ring vibration rather than C-H or N-H deformation. The resonance behavior (see below) of both the 1258- and 1331-cm-' peaks is consistent with assignment to ring modes. The three most intense Raman peaks of Cor11m'2 are observed a t 1163, 1273, and 1489 cm-l. In the ir spectrum of this complex, Cordes and Walter1* have assigned peaks a t 1160 and 1485 cm-I to ring vibrational modes and a peak a t 1273 cm-l to a combination band (948 340 cm-I). In the Raman spectrum, however, the peak a t 1273 cm-I is the most intense and, consequently, should be assigned to a fundamental vibration and not to a combination band. Resonance Effects. Albrecht and HutleyI9 have shown that when the incident radiation is near or within an absorption band of a molecule, the Raman scattering intensity of specific vibrations can be enhanced either through vibronic coupling with the single active excited electronic state in resonance (the A term of the vibronic theory) or through interaction of the major active state with electronic states a t higher energies coupled to it via the enhanced vibration (the B terms). For a diagonal element in the polarizability derivative tensor, the frequency dependence on incident radiation can be factored out of the A and B terms, giving frequency factors
+
F , = ~u'(v,v,
+
vo2)/(v;
-
u ~ ~ ) (-Y ~v O ~2)
(2)
where vo is the incident frequency, v = vo - Av, Av is the vibrational frequency, ve is the major active virtual electronic state for the vibrational mode a t Av, and vs is an average frequency for all other active virtual states assumed to lie a t much higher energy than ve. Vibrational intensities are proportional to FA^ or F B ~ . It is thus possible to obtain information on the source of the resonance enhancement of a given vibrational mode by comparing the observed intensity vs. wavelength dependence to that predicted from eq 1 and 2. These equations should be valid for the totally symmetric vibrations of the complexes studied. The observed and calculated relative intensity behavior is presented in Tables 11-IV. The Raman spectrum of imidazole was recorded with four different laser excitations (A 457.9, 476.5, 496.5, and Journal of the American Chemical Society
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-Wavelength of excitation, nm-. 457.9
488.0
514.5
1 .oo
1 .oo
1 .oo 1 .oo 1 .oo
0.92 1.11 1.09 1.04 1.11 0.94 1 .19 1.24
1.52 1.44 1.34 1 .25 1.36 1.45 1.41 1.48
1 .oo
I .16
1.35
1 .oo
1 .oo 1 .oo
Table 111. Normalized Raman Intensities of CoIIIm's Relative to 514.5-nm Excitation (see Table I1 and Text), and Calculated Values of FAI Raman freq, cm-' 1489 1273 1163 Calcd for 1000 cm-1. v e 395 nm v e 271 nm
-Wavelength of excitation, nm457.9
476,5
514.5
2.38 2.73 3.27
1.98 1.72 2.29
1 .oo 1 .oo 1 .oo
13.03 2.79
4.62 1.93
1 00 1 .oo
Table IV. Normalized Raman Intensities of C 0 ~ ~ 1 r n ~Relative Cl~ to 514.5-nm Excitation (see Table I1 and Text), and Calculated Values of FA^ Raman freq, cm-1 1534 1421 1322 1253 1165 1100 913 Calcd for 1000 cm-1, v e 425 nm v e 314n:n v e 250 nin
--Wavelength
of excitation, nm--
457.9
476.5
496.5
514.5
4.54 3.69 3.26 3.74 3.75 2.17 1.91
3.43 2.74 2.19 2.47 2.49 1.58 1.80
2.46 1.76 1.53 1.70 1.32 1.32 1.35
1.00 1.00 1.00 1.00 1.00 1.09 1.00
55.37 3.70 2.54
9.18 2.29 1.82
2.46 1.45 1.31
1.00 1.00 1.00
514.5 nm) to determine the vo dependence of peak intensities in free imidazole. All peak intensities remained unchanged relative to the V I vibration of S042-. Since the Raman spectra of pure aqueous imidazole show no excitation frequency dependent intensity variations, any variation observed in the spectra of the Co(I1) complexes must arise from an electronic state v, which involves the combined cobalt(I1)-imidazole moiety. Co111m6(N03)2.A slight dependence of peak intensities on excitation frequency is observed (Figure 2, Table 11) for Cor11m~(N03)2.The measured intensities increase as the excitation wavelength increases. That is, the electronic transition responsible for the intensity enhancement (v,) must lie a t the red end of the spectrum. The stoichiometry, visible absorption spectrum, and magnetic moment measurements indicate octahedral coordination of this complex.14 At the high wavelength end of the visible spectrum, the most intense absorption band is observed a t 1060 nm and has been assigned to the 4T1,(F) 4T2s(F) d-d transition of C0(II).I4 The frequency factor FA* calculated using the 1060-nm transition as the active electronic state is compared with the measured relative intensities in Table 11. The agreement between the calculated and measured intensities
March 5, 1975
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1031 tal intensity behavior. The poor fit between the calculated shows that this single term is quite successful in repreand experimental intensity ratios indicates that a single senting the experimentally observed wavelength dependence term cannot adequately represent the observed frequency of the imidazole peak intensities. dependence of the peak intensities. The actual peak intensiWe have observed a very intense absorption band with ties appear to result from the summation over several elecmaximum below 250 nm in the uv spectrum of an ethanolic tronic states of the tetrahedral-like complexes. solution of the complex. This transition and the 4Tlg(F) Metal-ligand charge transfer (CT) transitions are often 4Tls(P) d-d transition at 487 nm1,14however, appear to conthe active electronic states for resonance enhancement of tribute little to the observed peak intensity variation. Raman peak i n t e n ~ i t i e s . ~ IIn - ~these ~ two tetrahedral-like The slight resonance enhancement is reasonable in view complexes, a cobalt-imidazole C T transition or transition of the low energy of the 1060-nm band and reflects minimal near 250 nm appears to be the principal contributor to the mixing of this d-d state with ligand electronic levels at intensity variations while cobalt d-d transitions have no sighigher energy (
